While all the information necessary to encode the secondary and tertiary structure of a protein is contained in its linear sequence of amino acids, translation of this information from primary sequence to native structure often goes awry, resulting in protein mis-folding and aggregation. In some cases, aggregation of proteins can trigger severe cellular dysfunction and disease. Examples include cystic fibrosis, thalassemias, alpha1- antitrypsin deficiency, and several neuropathies such as Alzheimer's, Huntington's and Parkinson's diseases. The progression of these diseases is often correlated with the formation of protein fibrils. However, the growth and deposition of structured fibrils is generally preceded by the formation of amorphous and partially structured, pre-fibrillar states. Growing evidence suggests that pre-fibrillar, low-order aggregates play a central and common role in the pathology of many diseases. Importantly, protein aggregation is heavily influenced by the cellular protein quality control machinery, involving networks of molecular chaperones. Precisely how different chaperone systems cooperate to dismantle and reactivate aggregated proteins, and how molecular chaperone action affects disease progression, is not well understood. A significant impediment to a better understanding of protein aggregate disassembly by molecular chaperones is the inherently complex and heterogeneous nature of an aggregating protein sample. Aggregating proteins typically form a wide variety of conformational states and assemblies. This complex and broad distribution of states is, in general, very difficult to capture with current detection techniques. A principle goal of this proposal is to overcome this analytical limitation in order to develop a detailed mechanistic understanding of how an essential molecular chaperone network, consisting of ClpB, DnaKJ-GrpE and GroEL-ES, extracts and refolds proteins from aggregates. To accomplish this goal, we will: (1) develop a new analytical tool based on single-particle fluorescence burst detection that is capable of rapidly quantifying the specific molar distribution of states within an aggregated protein population, as well as how that distribution changes with time, (2) employ this method to examine the mechanism of protein aggregate disassembly by the DnaKJ-GrpE and ClpB bi-chaperone system, and (3) employ a combination of fluorescence spectroscopy and cryo-electron microscopy to determine how the binding of a non-native protein by DnaK, following extraction from an aggregate, affects the subsequent folding of the protein by GroEL.